Collagenase Description Life Technologies’ group of collagenase products are purified from Clostridium histolyticum. They are intended for cell and tissue disaggregation. Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-X- Gly-Pro. This sequence is found in high frequency in collagen. Collagenase is unique among proteases in its ability to degrade the triplehelical native collagen fibrils commonly found in connective tissue. The collagenase most commonly used for tissue dissociation is a crude preparation containing clostripiopeptidase A and a number of other proteases, polysaccharidases, and lipases. This crude preparation is ideally suited for tissue dissociation because it contains the enzyme required to attack native collagen and reticular fibers, in addition to the enzymes which hydrolyze the other proteins, polysaccharides, and lipids in the extracellular matrix of connective and epithelial tissues. Crude collagenase does exhibit lot-to-lot variability and may produce occasional toxicity. The activity of these crude collagenase preparations has been correlated with their effectiveness at dissociating specific tissue types leading to the classification of crude collagenase preparations by type. These selected types have been found to give better performance in preparation of cells from the various tissues (Table 1).

Product Catalog No. Amount Storage Collagenase: Type I 17100-017 1 g 2°C to 8°C Type II 17101-015 1 g 2°C to 8°C Type IV 17104-019 1 g 2°C to 8°C Collagenase, lyophilized 17018-029 500 mg 2°C to 8°C

Product Use Dissociate Tissue For Research Use Only. Not for use in diagnostic procedures 1. Mince tissue into 3-4 mm pieces with a sterile scalpel or scissors. Safety Information 2. Wash the tissue pieces several times with HBSS containing Read the Safety Data Sheets (SDSs) and follow the handling calcium and magnesium. instructions. Wear appropriate protective eyewear, clothing, and 3. Add sufficient HBSS with calcium and magnesium to gloves. Avoid inhalation and skin contact. submerge tissue. Add collagenase to 50–200 U/mL. Unit Definition 4. Incubate at 37°C for 4–18 hours. Increased efficiency is One protease unit liberates 1 µmol of L-leucine equivalents from obtained using a rocker platform and supplementing the collagen in 5 hours at 37°C, pH 7.5. digest with 3 mM CaCl2. 5. Disperse cells by passing through a sterile stainless steel or Table 1: Product Selection nylon mesh. Remaining tissue fragments may be Collagenase Tissue / Cell type disaggregated by addition to fresh collagenase solution and Type I Epithelial, Adrenal, Lung, Fat further incubation at 37°C. Type II Heart, Thyroid, Salivary, Liver, Bone, Cartilage 6. Wash dispersed cells several times by centrifugation in HBSS w/o collagenase. Type IV Islet (insulin receptor sites) 7. Resuspend cell pellet, after the final wash step, in culture medium. Determine viable cell density using a Countess® Use Automated Cell Counter (alternate automated or manual Reconstitute Collagenase methods may be used). 1. Add 1 mL Hank’s Balanced Salt Solution (HBSS) with 8. Seed cells into culture vessels containing appropriate media. calcium and magnesium directly to 1 g vial of Collagenase. Organ Perfusion Vortex gently to ensure complete dissolution. 1. Add collagenase to prewarmed (37°C) HBSS with calcium 2. Transfer to a clean tube. and magnesium. Addition of 3 mM CaCl2 increases the 3. Determine volume of HBSS with calcium and magnesium efficiency of dissociation. required to bring collagenase solution to 100 U/μL (1000X 2. Perfuse organ at preoptimized rate for the particular organ. stock solution). Rinse vial with this volume of HBSS with 3. Dispersed cells and tissue fragments are separated from calcium and magnesium, and combine. larger pieces by passing the perfusate through a sterile 4. Filter sterilize 1000X stock solution with a low protein stainless steel or nylon mesh. Remaining tissue fragments binding filtration unit. Use immediately or proceed to step 5. may be disaggregated by addition to fresh collagenase 5. Dispense into aliquots and store at –20°C to –5°C protected solution and further incubation at 37°C. from light. 4. Wash dispersed cells several times by centrifugation in 6. Thaw on ice prior to use. Avoid multiple freeze/thaw cycles. HBSS w/o collagenase. We recommend using collagenase at 50–200 U/mL concentration (or 0.1–0.5% W/V).

Publication Number MAN0007350 Rev. 1.00

5. Resuspend cell pellet, after the final wash step, in culture Related Products medium. Determine viable cell density using a Countess® Product Catalog No. Automated Cell Counter (alternate automated or manual methods may be used). HBSS, calcium, magnesium, no phenol red 14025 6. Seed cells into culture vessels containing appropriate media. Trypan Blue Stain 15250 Countess® Automated Cell Counter C10227 Explanation of Symbols and Warnings The symbols present on the product label are explained below:

Manufacturer Catalog number Batch code

Caution, consult Consult instructions Temperature accompanying documents for use Limitation Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.

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